DIXDC1 Promotes Retinoic Acid-Induced Neuronal Differentiation and Inhibits Gliogenesis in P19 Cells

Human DIXDC1 is a member of Dishevelled-Axin (DIX) domain containing gene family which plays important roles in Wnt signaling and neural development. In this report, we first confirmed that expression of Ccd1, a mouse homologous gene of DIXDC1, was up-regulated in embryonic developing nervous system. Further studies showed that Ccd1 was expressed specifically in neurons and colocalized with early neuronal marker Tuj1. During the aggregation induced by RA and neuronal differentiation of embryonic carcinoma P19 cells, expressions of Ccd1 as well as Wnt-1 and N-cadherin were dramatically increased. Stable overexpression of DIXDC1 in P19 cells promoted the neuronal differentiation. P19 cells overexpressing DIXDC1 but not the control P19 cells could differentiate into Tuj1 positive cells with RA induction for only 2 days. Meanwhile, we also found that overexpression of DIXDC1 facilitated the expression of Wnt1 and bHLHs during aggregation and differentiation, respectively, while inhibited gliogenesis by down-regulating the expression of GFAP in P19 cells. Thus, our finding suggested that DIXDC1 might play an important role during neurogenesis, overexpression of DIXDC1 in embryonic carcinoma P19 cells promoted neuronal differentiation, and inhibited gliogenesis induced by retinoic acid. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. XT Jing and HT Wu contributed equally to this work.     

A role of N-cadherin in neuronal differentiation of embryonic carcinoma P19 cells.

N-cadherin is one of the important molecules for cell to cell interaction in the development of the central nervous system (CNS). In this report, we have shown that N-cadherin mRNA and protein were increased rapidly in retinoic acid (RA)-induced neuronal differentiation of embryonic carcinoma P19 cells. To explore possible roles for N-cadherin during this process, N-cadherin-overexpressing P19 cell lines were established. These transfected cells could differentiate into neurofilament-expressing neurons in the absence of RA. RT-PCR revealed that the expression patterns of development-related genes, such as Oct-3/4, nestin, Notch-1, and Mash-1 were similar between the transfected P19 cells and the RA-induced wild-type P19 cells during their neuronal differentiation. On the contrary, the Wnt-1 gene was up-regulated in the N-cadherin-overexpressing P19 cells, but could not be detected in the wild-type P19 cells. These results suggest N-cadherin may play a role in neuronal differentiation of P19 cells, possibly through the Wnt-1 signaling pathway.     

Expression analysis of Fgf8a & Fgf8b in early stage of P19 cells during neural differentiation

Fgf8 is a member of the fibroblast growth factor (FGF) family that plays an important role in early neural development. Cellular aggregation and retinoic acid (RA) are needed for mouse embryonic carcinoma (EC) P19 cell neural differentiation. We have examined the Fgf8 gene in P19 cells during neural differentiation and identified 2 alternatively spliced Fgf8 isoforms, Fgf8a and Fgf8b, among the 8 known splicing isoforms in mammals. The expression of Fgf8a and Fgf8b mRNAs transiently and rapidly increased in the early stage of P19 cells during RA-induced neural differentiation, followed by a decline in expression. The relative amount of Fgf8b was clearly higher than that of Fgf8a at different time-points measured within 24 h after RA treatment. Increased Fgf8b mRNA expression was cellular-aggregation dependent. The results demonstrated that cellular-aggregation-induced Fgf8b, but not Fgf8a, may play a pivotal role in early neural differentiation of P19 cells.     

Directed differentiation of mouse P19 embryonal carcinoma cells to neural cells in a serum- and retinoic acid-free culture medium

P19 embryonal carcinoma cells (EC-cells) provide a simple and robust culture system for studying neural development. Most protocols developed so far for directing neural differentiation of P19 cells depend on the use of culture medium supplemented with retinoic acid (RA) and serum, which has an undefined composition. Hence, such protocols are not suitable for many molecular studies. In this study, we achieved neural differentiation of P19 cells in a serum- and RA-free culture medium by employing the knockout serum replacement (KSR) supplement. In the KSR-containing medium, P19 cells underwent predominant differentiation into neural lineage and by day 12 of culture, neural cells were present in 100% of P19-derived embryoid bodies (EBs). This was consistently accompanied by the increased expression of various neural lineage-associated markers during the course of differentiation. P19-derived neural cells comprised of NES⁺ neural progenitors (~?46%), TUBB3⁺ immature neurons (~?6%), MAP2⁺ mature neurons (~?2%), and GFAP⁺ astrocytes (~?50%). A heterogeneous neuronal population consisting of glutamatergic, GABAergic, serotonergic, and dopaminergic neurons was generated. Taken together, our study shows that the KSR medium is suitable for the differentiation of P19 cells to neural lineage without requiring additional (serum and RA) supplements. This stem cell differentiation system could be utilized for gaining mechanistic insights into neural differentiation and for identifying potential neuroactive compounds.     

NELL2 promotes motor and sensory neuron differentiation and stimulates mitogenesis in DRG in vivo

We previously identified a secreted glycoprotein, neural epidermal growth factor-like like 2 (NELL2), in a subtraction screen designed to identify molecules regulating sensory neurogenesis and differentiation in the chick dorsal root ganglion (DRG). Characterization of NELL2 expression during embryogenesis revealed that NELL2 was specifically expressed during the peak periods of both sensory and motor neuron differentiation, and within the neural crest was restricted to the sensory lineage. We now provide evidence for a function for NELL2 during neuronal development. We report here that NELL2 acts cell autonomously within CNS and PNS progenitors, in vivo, to promote their differentiation into neurons. Additionally, neuron-secreted NELL2 acts paracrinely to stimulate the mitogenesis of adjacent cells within the nascent DRG. These studies implicate dual functions for NELL2 in both the cell autonomous differentiation of neural progenitor cells while simultaneously exerting paracrine proliferative activity.     

Methyltransferase-inhibition interferes with neuronal differentiation of P19 embryonal carcinoma cells

We have analyzed the importance of substrate methylation by S-adenosylmethionine-dependent methyltransferases for neuronal differentiation of P19 embryonal carcinoma cells. We show that treatment of cells with methyltransferase inhibitor adenosine dialdehyde (AdOx) interferes with neuronal differentiation. Retinoic acid (RA) and AdOx co-treated cells had a decreased number of neurites and a flattened morphology compared with cells differentiated by RA. Also, the amount of neuronal class III tubulin (Tuj1) decreased from 76% to 9.6% with AdOx-treatment. Gene expression levels of wnt-1, brn-2, neuroD, and mash-1 were also down-regulated by AdOx-treatment. But AdOx-treatment did not up-regulate BMP-4 and GFAP genes. Treatment of RA decreased E-cadherin expression during neuronal differentiation. However, in AdOx/RA co-treated cells, E-cadherin expression was restored to the control level. Also, mRNA expression of N-cadherin decreased with AdOx-treatment. Taken together, these data show that methylation reactions might influence the cell-fate decision and neuronal differentiation of P19 cells.     

ADAM23 knockdown promotes neuronal differentiation of P19 embryonal carcinoma cells by up‐regulating P27KIP1 expression

ADAM23 (a disintegrin and metalloprotease 23), a member of brain MDC (macrophage‐derived chemokine) family, is important for the development of CNS (central nervous system). P19 mouse embryonal carcinoma cells can differentiate into neurons when cultured in aggregates and induced with RA (retinoic acid). We have found that under conditions without RA induction, knocking down ADAM23 with RNAi (RNA interference) promoted neuronal differentiation, and similarly recombinant GST (glutathione transferase)‐ADAM23‐DIS protein inhibited neuronal differentiation of P19/ADAM23KD (P19/ADAM23‐knockdown) cells. In P19/ADAM23KD, there were more cells arrested in G1 phase than normal P19 cells, due to the up‐regulation of P57^KIP2 and P27^KIP1 expression. P27^KIP1 was up‐regulated during the differentiation process of both P19/ADAM23KD cells without RA induction, and P19 cells with RA induction. Transient overexpression of P27^KIP1 in P19 cells also promoted neuronal differentiation of P19 cells. The findings indicate that ADAM23 suppresses neuronal differentiation through its disintegrin domain, and Adam23 KD up‐regulates P27^KIP1 in P19/ADAM23KD cells, one reason that P19/ADAM23KD cells can differentiate into neurons without RA induction.     

RBM4 promotes neuronal differentiation and neurite outgrowth by modulating Numb isoform expression

RBM4 participates in cell differentiation by regulating tissue-specific alternative pre-mRNA splicing. RBM4 also has been implicated in neurogenesis in the mouse embryonic brain. Using mouse embryonal carcinoma P19 cells as a neural differentiation model, we observed a temporal correlation between RBM4 expression and a change in splicing isoforms of Numb, a cell-fate determination gene. Knockdown of RBM4 affected the inclusion/exclusion of exons 3 and 9 of Numb in P19 cells. RBM4-deficient embryonic mouse brain also exhibited aberrant splicing of Numb pre-mRNA. Using a splicing reporter minigene assay, we demonstrated that RBM4 promoted exon 3 inclusion and exon 9 exclusion. Moreover, we found that RBM4 depletion reduced the expression of the proneural gene Mash1, and such reduction was reversed by an RBM4-induced Numb isoform containing exon 3 but lacking exon 9. Accordingly, induction of ectopic RBM4 expression in neuronal progenitor cells increased Mash1 expression and promoted cell differentiation. Finally, we found that RBM4 was also essential for neurite outgrowth from cortical neurons in vitro. Neurite outgrowth defects of RBM4-depleted neurons were rescued by RBM4-induced exon 9–lacking Numb isoforms. Therefore our findings indicate that RBM4 modulates exon selection of Numb to generate isoforms that promote neuronal cell differentiation and neurite outgrowth.     

Retinoic acid treatment and cell aggregation independently regulate alternative splicing in P19 cells during neural differentiation

To induce neural differentiation of P19 cells, two different treatments, RA (retinoic acid) and cell aggregation, are required. However, there has been no report that RA treatment alone or cell aggregation alone could control alternative splicing regulation in P19 cells. Therefore, we focused on alternative splicing effects by neural induction (RA treatment and/or cell aggregation) in P19 cells. We analysed the splicing patterns of several genes, including 5‐HT3R‐A (5‐hydroxytryptamine receptor), Actn1 (actinin alpha1), CUGBP2 (CUG‐binding protein) and PTB (polypyrimidine track‐binding protein), which showed different responses during the early neural induction of P19 cells. We show here that RA treatment alone changes the alternative splice mechanism of 5‐HT3R‐A. Cell aggregation alone controls alternative splicing regulation of Actn1. Both treatments (RA and cell aggregation) compensate and regulate the alternative splicing mechanism of CUGBP2. However, PTB is independent of RA and cell aggregation. Taken together, our results suggest that RA treatment and cell aggregation independently regulate the alternative splicing mechanism in the early stage of P19 cells during neural differentiation.     

Restricted neural epidermal growth factor-like like 2 (NELL2) expression during muscle and neuronal differentiation

We have identified a secreted glycoprotein, neural epidermal growth factor-like like 2 (NELL2), in a screen designed to isolate molecules regulating sensory neuron genesis and differentiation in the dorsal root ganglia (DRG). In investigating NELL2 expression during embryogenesis, we demonstrate here that NELL2 is highly regulated spatially and temporally, being only transiently expressed in discrete regions of the central (CNS) and peripheral nervous systems (PNS) and in a subset of mesoderm derived structures during their peak periods of development. In the CNS and PNS, NELL2 is maximally expressed as motor and sensory neurons differentiate. Interestingly, its expression is restricted to sublineages of the neural crest, being strongly expressed throughout the immature DRG, but excluded from sympathetic ganglia. Similarly during muscle development, NELL2 is specifically expressed by hypaxial muscle precursor cells in the differentiating somite and derivatives in the forelimbs and body wall, but not by epaxial muscle precursors. Furthermore, NELL2 is differentially regulated in the CNS and PNS; in the CNS, NELL2 is only expressed by nascent, post-mitotic neurons as they commence their differentiation, yet in the PNS, NELL2 is expressed by subsets of progenitor cells in addition to nascent neurons. Based on this restricted spatial and temporal expression pattern, functional studies are in progress to determine NELL2's role during neuronal differentiation in both the PNS and CNS.     

pp60src tyrosine kinase modulates P19 embryonal carcinoma cell fate by inhibiting neuronal but not epithelial differentiation

P19 embryonal carcinoma cells provide an in vitro model system to analyze the events involved in neural differentiation. These multipotential stem cells can be induced by retinoic acid (RA) to differentiate into neural cells. We have investigated the ability of several variant forms of the protein-tyrosine kinase (PTK) pp60src to modulate cell fate determination in this system. Normally, P19 cells are induced to differentiate along a neural lineage when allowed to form extensive cell-cell contacts in large multicellular aggregates during exposure to RA. Through analysis of markers of epithelial (keratin and desmosomal proteins) and neuronal (neurofilament) cells we have found that RA-induced P19 cells transiently express epithelial markers before neuronal differentiation. Under these inductive conditions, expression of pp60v-src or expression of the neuronal variant pp60c-src+ inhibited neuronal differentiation, and resulted in maintained expression of an epithelial phenotype. Morphological analysis showed that expression of pp60src PTKs results in decreased cell-cell adhesion during the critical cell aggregation stage of the neural differentiation procedure. The effects of pp60v-src on cell fate and cell-cell adhesion could be mimicked by direct modulation of Ca+(+)-dependent cell-cell contact during RA induction of normal P19 cells. We conclude that the neural lineage of P19 cells includes an early epithelial intermediate and suggest that tyrosine phosphorylation can modulate cell fate determination during an early cell-cell adhesion-dependent event in neurogenesis.     

Restricted neural epidermal growth factor-like like 2 (NELL2) expression during muscle and neuronal differentiation

We have identified a secreted glycoprotein, neural epidermal growth factor-like like 2 (NELL2), in a screen designed to isolate molecules regulating sensory neuron genesis and differentiation in the dorsal root ganglia (DRG). In investigating NELL2 expression during embryogenesis, we demonstrate here that NELL2 is highly regulated spatially and temporally, being only transiently expressed in discrete regions of the central (CNS) and peripheral nervous systems (PNS) and in a subset of mesoderm derived structures during their peak periods of development. In the CNS and PNS, NELL2 is maximally expressed as motor and sensory neurons differentiate. Interestingly, its expression is restricted to sublineages of the neural crest, being strongly expressed throughout the immature DRG, but excluded from sympathetic ganglia. Similarly during muscle development, NELL2 is specifically expressed by hypaxial muscle precursor cells in the differentiating somite and derivatives in the forelimbs and body wall, but not by epaxial muscle precursors. Furthermore, NELL2 is differentially regulated in the CNS and PNS; in the CNS, NELL2 is only expressed by nascent, post-mitotic neurons as they commence their differentiation, yet in the PNS, NELL2 is expressed by subsets of progenitor cells in addition to nascent neurons. Based on this restricted spatial and temporal expression pattern, functional studies are in progress to determine NELL2's role during neuronal differentiation in both the PNS and CNS.     

Involvement of Crosstalk between Oct4 and Meis1a in Neural Cell Fate Decision

Oct4 plays a critical role both in maintaining pluripotency and the cell fate decision of embryonic stem (ES) cells. Nonetheless, in the determination of the neuroectoderm (NE) from ES cells, the detailed regulation mechanism of the Oct4 gene expression is poorly understood. Here, we report that crosstalk between Oct4 and Meis1a, a Pbx-related homeobox protein, is required for neural differentiation of mouse P19 embryonic carcinoma (EC) cells induced by retinoic acid (RA). During neural differentiation, Oct4 expression was transiently enhanced during 6–12 h of RA addition and subsequently disappeared within 48 h. Coinciding with up-regulation of Oct4 expression, the induction of Meis1a expression was initiated and reached a plateau at 48 h, suggesting that transiently induced Oct4 activates Meis1a expression and the up-regulated Meis1a then suppresses Oct4 expression. Chromatin immunoprecipitation (ChIP) and luciferase reporter analysis showed that Oct4 enhanced Meis1a expression via direct binding to the Meis1 promoter accompanying histone H3 acetylation and appearance of 5-hydoxymethylcytosine (5hmC), while Meis1a suppressed Oct4 expression via direct association with the Oct4 promoter together with histone deacetylase 1 (HDAC1). Furthermore, ectopic Meis1a expression promoted neural differentiation via formation of large neurospheres that expressed Nestin, GLAST, BLBP and Sox1 as neural stem cell (NSC)/neural progenitor markers, whereas its down-regulation generated small neurospheres and repressed neural differentiation. Thus, these results imply that crosstalk between Oct4 and Meis1a on mutual gene expressions is essential for the determination of NE from EC cells.     

Biochemical characterization and expression analysis of neural thrombospondin-1-like proteins NELL1 and NELL2.

Two closely related genes coding for NELL proteins (NELL1 and NELL2) have been cloned by the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C betaI (PKCbetaI) as bait. The rat NELL proteins show about 55% identity with each other and contain several protein motifs assigned to a secretion signal peptide, an NH(2)-terminal thrombospondin-1 (TSP-1)-like module, five von Willebrand factor C domains, and six epidermal growth factor-like domains; the NELL proteins share many protein motifs with TSP-1. The NELL proteins expressed in COS-7 cells are homotrimeric glycoproteins and possess heparin-binding activity. Furthermore, while NELL1 and NELL2 show distinct subcellular localization in cytoplasm, they both are partially secreted into the culture medium of COS-7 cells. Although the NELL1 mRNA is faintly expressed in adult neural cells, the NELL2 mRNA is expressed abundantly, particularly in the pyramidal cells of rat hippocampus, showing neuronal high plasticity. During mouse embryogenesis, expression of the NELL2 mRNA is initiated 7-11 days postcoitum, simultaneously with neural plate formation. These results strongly suggest that the NELL2 protein, similar to but not identical with TSP-1, is involved in the growth and differentiation of neural cells. Additionally, the NELL1 and NELL2 mRNAs were found to be expressed abundantly in Burkitt's lymphoma Raji cells and colorectal adenocarcinoma SW480 cells, respectively. Thus, it is likely that the NELL proteins also participate in the growth, differentiation, and oncogenesis of cancer cell lines.